Crystal Structure, SAXS and Kinetic Mechanism of Hyperthermophilic ADP-Dependent Glucokinase from Thermococcus litoralis Reveal a Conserved Mechanism for Catalysis

Abstract

ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Termococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.

Both protein dynamics and ligand concentration can shift the binding mechanism between conformational selection and induced fit

“…Generally, significant differences in protein conformation exist between the unbound and bound states, as exemplified by hemoglobin upon binding oxygen (1)(2)(3)(4) and HIV-1 protease upon binding a substrate or a drug molecule (5). In the latter as well as some other cases (6)(7)(8)(9)(10), loops and other groups collapse around the bound ligand, leading to a closed binding pocket. The conformational redistribution and dynamics of the protein molecule exhibited during the binding process can potentially play a critical role in determining the magnitude of the rate constant as well as the mechanism of ligand binding (11,12).…”

“…However, detractors of CS have noted that, at least for cases with a closed binding pocket in the active conformation, direct binding to the latter conformation cannot proceed (9,15). In some cases, a partially closed conformation has been observed by a sensitive probe such as paramagnetic relaxation enhancement (20) or in molecular dynamics simulations.…”

The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase

“…Archaeal ADPGK crystal structures have been determined for apo-ADPGK from Pyrococcus horikoshii OT3 (PDB code 1L2l) (7) and glucose-and AMP-bound Pyrococcus furiosus ADPGK (PDB code 1UA4) (8). Thermococcus litoralis ADPGK structures have been determined in the ADPbound form (PDB code 1GC5) (9) and apo (PDB code 4B8R) and AMP/glucose ternary complexes (PDB code 4B8S) (10). The ADPGK structure contains two ␣/␤ domains; the large domain is a Rossmann-type fold of an eight-stranded ␤-sheet enclosed by eight ␣-helices, with five helices on one side and three on the other.…”

“…The different archaeal ADPGK liganded states are consistent with an induced fit ligand-binding model involving a hinged rigid body interdomain conformational change with a domain closing movement over the ADP and glucose once bound in the active site. Kinetic studies of T. litoralis ADPGK have led to a proposed ordered sequential enzymatic mechanism with MgADP binding before D-glucose (10). This mechanism has been rationalized with respect to the observed structural states, although SAXS data suggest that the conformational change in solution is greater than that seen by the ADPGK crystal structures (10).…”

“…Kinetic studies of T. litoralis ADPGK have led to a proposed ordered sequential enzymatic mechanism with MgADP binding before D-glucose (10). This mechanism has been rationalized with respect to the observed structural states, although SAXS data suggest that the conformational change in solution is greater than that seen by the ADPGK crystal structures (10). For archaeal ADPGKs, the specificity for ADP over ATP has been explained by amino acid side chains within the nucleotide binding site positioning the ADP ␤-phosphate in an homologous position to the ␥-phosphate of ATP-dependent kinases (9) for phosphoryl transfer.…”